CN105617461A - Skin patch for treating skin burn and preparation method of skin patch - Google Patents
Skin patch for treating skin burn and preparation method of skin patch Download PDFInfo
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Abstract
The invention relates to a preparation method of a skin patch for treating a skin burn. The preparation method comprises the following steps: respectively obtaining adipose-derived stem cells and allogenic skin matrix; mixing the adipose-derived stem cells with a collagen solution and then inoculating into the allogenic skin matrix, wherein the adipose-derived stem cells are from a patient with the skin burn. By differentiation potential of the adipose-derived stem cells and the stimulation-inducing effect of the allogenic skin matrix, the adipose-derived stem cells are prompted to differentiate into epidermis cells. The adipose-derived stem cells are from the patient with the skin burn, so that the antigenicity is relatively low, and immunological rejection is not easily caused, therefore, the skin repair effect is improved; the target of quickly repairing the skin wound is achieved. In addition, the repairing speed is increased, so that pigment accumulation of the wound part is not easily caused to form a scar and the like; and the pain of the patient is relieved.
Description
Technical field
The present invention relates to field of tissue engineering technology, particularly to a kind of for transdermal patches treating skin burn and preparation method thereof.
Background technology
Skin is the barrier that human body contacts with external environment, not only plays the effects such as protection, secretion, metabolism and sensation, and participates in immunoreation and maintain stablizing of organismic internal environment. As the skin that human body is showed most, it is impaired injures large area to scald unexpected incidence rate of burning higher. Early stage burn wound cover can bacteria growing inhibiting, prevention infect, prevent moisture and electrolytical loss; Auto-skin grafting is the first-selection for the treatment of burn, but for large-area burns, autologous skin can not meet demand, and the cycle course for the treatment of is longer, and in therapeutic process, infecting easily occurs in skin wounds; Allograft skin substrate skin then can cause obvious immunological rejection; And transplant artificial skin, subcutaneous secretions is easily formed vesicle, and then artificial skin causes damage, and artificial skin is costly, and processing technology is more complicated. Therefore, so far, the skin source of severe trauma and Patients with Big Area Burn lacks the difficult problem remaining clinical urgently to be resolved hurrily.
At present, the research of tissue engineering skin has become one of the focus in life science field, and market has also occurred for clinical organization engineering skin product, though there being certain clinical effectiveness, but it is not only expensive, technical operation is loaded down with trivial details, has certain technical difficulty and cost higher.
Summary of the invention
The technical problem to be solved is, immunological rejection and the defect such as curative effect efficiency is low is easily caused, it is provided that a kind of it can be avoided that immunological rejection and repairing effect are preferably for transdermal patches treating skin burn and preparation method thereof for prior art adopting allograft skin substrate repair skin burn wound.
The technical solution adopted for the present invention to solve the technical problems is: the preparation method providing a kind of transdermal patches for treating skin burn, comprises the following steps:
Obtain fat stem cell and allograft skin substrate respectively;
Described allograft skin substrate it is inoculated in collagen solution after being mixed by described fat stem cell;
Wherein, described fat stem cell derives from skin burn autologous patient.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, final concentration of (2��4) �� 10 of described fat stem cell4Individual/ml.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, in described collagen solution, the mass fraction of collagen is 0.1��0.3%.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, the acquisition process of described fat stem cell, comprise the following steps:
Under aseptic condition, take the fatty tissue of described skin burn patient, reject macroscopic blood vessel and fiber part in fat, after PBS solution is cleaned, shred, enzymolysis vibration digestion 20��60 minutes, stop digestion with complete medium, then be placed in ice bath and blow and beat digestion 10��20 minutes, filter, after removing indigested fatty tissue and connective tissue, filtrate is centrifuged, removes upper strata oil droplet, take off a layer adipose cell, after PBS, namely obtain primary fat stem cell.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, the acquisition process of described fat stem cell is further comprising the steps of:
Wash described primary fat stem cell, remove erythrocyte, resuspended with complete medium, then it is inoculated on the coated culture plate of poly-ornithine, cultivate 2��6 hours, remove trypsinization after supernatant, remove indigested fibroblast and endotheliocyte, namely obtain the primary fat stem cell after purification.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, the acquisition process of described fat stem cell is further comprising the steps of:
Take the primary fat stem cell after purification with 0.5��2 �� 105The density of individual/ml is inoculated in DMEM culture medium and carries out amplification cultivation 7��10 days, changes liquid every 2-3 days once.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, described DMEM culture medium is added with the LIF of EGF and the 5��15ng/ml of 2��5% human serums, the ITS of 5��15ng/ml, the streptomycin of 50��150U/ml, 50��150U/ml penicillin, 5��35ng/ml.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, the acquisition process of described allograft skin substrate, comprise the following steps:
Take health pig abdominal part or back skin carries out defat depilation process, remove the epidermis of 0.2��0.6mm, make the skin corium skin graft that thickness is 0.2��0.4mm, sterilize 20��40 minutes after cleaning, repeatedly clean, digest 2��4 days in Digestive system, repeatedly clean skin graft, be subsequently adding nonionic surfactant and hatch 2��4 days, remove completely to cell component, obtain acellular dermal, then repeatedly rinse with sodium chloride and PBS respectively, namely obtain allograft skin substrate.
In the preparation method of the transdermal patches for treating skin burn provided by the invention, including mass percent in described Digestive system is 0.10��0.40% trypsin, 0.01��0.03% ethanedioic acid ethylenediamine and 0.01��0.03% sodium hydroxide.
The present invention also provides for a kind of transdermal patches for treating skin burn, the above-mentioned transdermal patches preparation method for treating skin burn obtain.
Implement provided by the invention for transdermal patches treating skin burn and preparation method thereof, following beneficial effect can be reached: by utilizing the differentiation potential of fat stem cell, and the stimulation inducing action of allograft skin substrate, promote fat stem cell to be divided into epidermis cell; And due to fat stem cell, to derive from fire victim autologous, and therefore its antigenicity is relatively low, not easily causes immunological rejection, thus improving skin repair effect, reaches quickly to repair the purpose of skin trauma; In addition, owing to accelerating reparation speed, therefore do not easily cause the accumulation of wound site pigment and form cicatrix etc., alleviate patient suffering.
Detailed description of the invention
Exist easily cause immunological rejection and the defect such as remediation efficiency is low for solving prior art repairs burned skin by allograft skin substrate, the innovative point of the present invention is in that to provide a kind of transdermal patches being loaded with skin burn autologous patient fat stem cell and preparation method thereof, by adopting autologous fat stem cell in conjunction with allograft skin substrate, to reduce the immunological rejection because allograft skin substrate causes, thus reducing the antigenicity of transdermal patches, improve skin repair efficiency.
Specifically, the transdermal patches for treating skin burn provided by the invention, its preparation method comprises the following steps:
S1, respectively acquisition fat stem cell and allograft skin substrate;
Wherein, fat stem cell derives from skin burn autologous patient; Allograft skin substrate is the dermal matrix after de-cell processes, it is possible to be alloskin substrate (namely allowancing for bark the mankind's scytoblastema matter beyond skin fire victim), it is also possible to be heterogenous allosome scytoblastema matter (such as the animal skins substrate such as pig, cattle);
S2, fat stem cell is mixed with collagen solution after be inoculated in allograft skin substrate.
Allograft skin substrate is not only used to as the paster repairing burned skin, and wherein remain the insoluble components in original organizational structure, mainly include collagen, elastin laminin, glycosaminoglycan and structural protein etc., provide Basal nutrition for Growth of Cells and skin ultrastructure to provide growth desired nutritional material for fat stem cell and promote fat stem cell to be divided into the cytokine of epidermis cell, make fat stem cell be able to maintain that its cytoactive, and epidermis cell can be divided into; And owing to fat stem cell derives from burned skin patient's body, therefore its antigenicity is relatively weak, it is possible to avoid allograft skin substrate directly to contact the immunological rejection caused by skin burn part, improve, with this, the purpose repairing skin efficiency.
Further, in step S1, it is preferable that the acquisition process of fat stem cell, comprise the following steps:
S11A, primary fat stem cell acquisition;
Under aseptic condition, take the fatty tissue of described skin burn patient, reject macroscopic blood vessel and fiber part in fat, after PBS solution is cleaned, shred, enzymolysis vibration digestion 20��60 minutes, stop digestion with complete medium, then be placed in ice bath and blow and beat digestion 10��20 minutes, filter, after removing indigested fatty tissue and connective tissue, filtrate is centrifuged, removes upper strata oil droplet, take off a layer adipose cell, after PBS, namely obtain primary fat stem cell;
Wherein it is preferred to, complete medium is the DMEM in high glucose culture medium dual anti-containing (5��10) % hyclone and (0.5��2) %.
S12A, in step S11A obtain primary fat stem cell be purified;
The primary fat stem cell obtained in washing step S11A, remove erythrocyte, resuspended with complete medium, then it is inoculated on the coated culture plate of poly-ornithine, cultivate 2��6 hours, remove trypsinization after supernatant, remove indigested fibroblast and endotheliocyte, namely obtain the primary fat stem cell after purification.
S13A, the primary fat stem cell after purification is carried out amplification cultivation;
Take in step S12A the primary fat stem cell after purification with (0.5��2) �� 105The density of individual/ml is inoculated in DMEM culture medium to carry out amplification cultivation 7��10 days, changed liquid every 2-3 days once. In the present invention, it is preferred to, primary fat stem cell is inoculated in cell culture bags, it is simple to post incoulation is coated in allograft skin substrate; Simultaneously in primary fat stem cell incubation, it is more beneficial for primary fat stem cell by weak vibrations cell culture bags and is fully contacted culture medium, to improve fat stem cell activity.
Wherein, DMEM culture medium is added with the EGF (EpidermalGrowthFactor of 2��5% human serums, the ITS (its Main Ingredients and Appearance is insulin, transferrins and selenium) of 5��15ng/ml, the streptomycin of 50��150U/ml, 50��150U/ml penicillin, 5��35ng/ml, epidermal growth factor) and the LIF (leukemiainhibitoryfactor, leukaemia inhibitory factor) of 5��15ng/ml.
Wherein, the insulin in ITS, it is possible to reduce protein degradation, increase the absorption of aminoacid and glucose, provide energy for Growth of Cells and differentiation, promote fat stem cell to be in low sugar environment, keep cytoactive, be conducive to fat stem cell to break up to epidermis cell; Meanwhile, it is capable to promote cells use glucose and protein, promote the synthesis of RNA, protein and fatty acid, it is suppressed that apoptosis, is very important liability factor. Transferrins can intervene the ion metabolism in fat stem cell, promotes the generation of fat stem cell. And selenium is powerful anti-oxidizing elements, selenium eliminates the free radical produced in cell cultivation process by polyphenoils, reduces and delay the formation of lipofuscin, to reach the purpose of anti-cell aging and apoptosis; It addition, selenium is as the composition of glutathion peroxidase, can catalysis reduced glutathion be oxidized form of glutathione, protect the biofilm structure of cell and the integrity of function, and protect cell membrane function and prevention necrocytosis and sudden change.
Penicillin can hinder the Cell wall synthesis of antibacterial, causes that cellular leakage is dead, thus suppressing the growth of antibacterial; And streptomycin can act on the ribosome of antibacterial, hinder protein translation, thus bacteria growing inhibiting; Under natural environment, being left out the Drug resistance of artificial generation, most microorganisms that penicillin is insensitive are sensitive to streptomycin, and vice versa; Therefore, use of most preferably penicillin and streptomycin being arranged in pairs or groups can control almost all common antibacterial such that it is able to avoid the harmful effect that growth and the activity of fat stem cell are caused by antibacterial as far as possible.
EGF can promote the migration of cell, stick, breeds and survive, and EGF can the propagation of induced lipolysis stem cell, migration, and do not affect its differentiation potential, damage tissue had reparation and defencive function.
LIF can promote Growth of Cells, propagation and differentiation, cell culture medium adds a certain amount of LIF and can replace trophoblastic cell, maintain the propagation of fat stem cell, improve cell proliferation rate and long-term proliferative capacity, and make fat stem cell can keep undifferentiated state.
It is understood that in the present invention, it is possible to directly adopt primary fat stem cell to make transdermal patches.
Further, the acquisition process of allograft skin substrate is:
S1B, take health pig abdominal part or back skin carries out defat depilation process, remove the epidermis of 0.2��0.6mm, make the skin corium skin graft that thickness is 0.2��0.4mm, sterilize 20��40 minutes after cleaning, repeatedly clean, digest 2��4 days in Digestive system, repeatedly clean skin graft, be subsequently adding nonionic surfactant and hatch 2��4 days, remove completely to cell component, obtain acellular dermal, then repeatedly rinse with sodium chloride solution and PBS respectively, fully to reduce the antigenicity of scytoblastema matter; Wherein, the mass fraction of sodium chloride is 5��9%, is 8��16 hours with the sodium chloride solution flushing process time, changed liquid every 6 hours; PBS flushing processes 8��16 hours, changes liquid every 6 hours, namely obtains allograft skin substrate.
Wherein it is preferred to, including mass percent in Digestive system is 0.10��0.40% trypsin, 0.01��0.03% ethanedioic acid ethylenediamine and 0.01��0.03% sodium hydroxide; Nonionic surfactant is (0.4��0.6) %TritonX-100 solution.
In step 2, before inoculation fat stem cell, allograft skin substrate need to being carried out sterilization processing, to reduce the antigenicity of allograft skin substrate further, detailed process is:
Under gnotobasis, taking the allograft skin substrate obtained in step S1B and load in sterile aluminum foil film, vacuum-pumping and sealing, with 60Coradiation mycete 3��5 hours; Wherein, irradiation dose is 3��5kGy, and the physicochemical property not change allograft skin substrate is advisable.
Additionally, before inoculation fat stem cell, also need in gnotobasis, allograft skin substrate after sterilization is paved, and allograft skin substrate epithelial surface is upward, specifically can allograft skin substrate be fixed is laid on the glass plate being slightly less than allograft skin substrate area, or tile and in Tissue Culture Dish, unnecessary allograft skin substrate infolding, the inoculating surfaces making allograft skin substrate is in formation state, then fat stem cell is inoculated, and after inoculation fat stem cell, allograft skin substrate need to remain formation state so that fat stem cell is uniformly distributed in allograft skin substrate, fat stem cell is avoided to pile up, affect its cytoactive and normal growth.
The detailed process of step S2 is:
Take the collagen solution that mass fraction is 0.1��0.3%, adjust PH to 7.0��7.4, the fat stem cell obtained and collagen solution are quickly sufficiently mixed, make final concentration of (2��4) �� 10 of fat stem cell in step S13A4Individual/ml;
Then, entering the collagen solution containing fat stem cell on allograft skin substrate upper berth, height 1��3mm, room temperature stands 10��30min to collagen set.
In order to make the purpose of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated. Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1
A kind of transdermal patches for treating skin burn provided by the invention, its preparation method comprises the following steps:
S1a, patient select;
Burn patient inclusion criteria: II��III degree of burn wound patient, side by side divided by lower patient:
Conceived women breast-feeding their children; To biological preparation allergy sufferers used in treatment; Moderate and severe inhalation and the damage of other important organs, as: hepatic and renal function damage, craniocerebral injury etc.; Serious underlying disease such as diabetes, hypertension, tumor or chronic organ injury etc.; Hinder signature and understand informed consent person.
Before S2a, treatment, patient is carried out inspection and evaluation;
Evaluation criteria: inspection routine blood test (WBC > 3.0 �� 10*9/L, LC > 15%; Hb > 80g/L; PLT > 60 �� 10*9/L), hepatic and renal function, hepatitis B three be that (surface antigen negative), c-hepatitis antibody feminine gender, AIDS negative antibody, syphilis antibody are negative.
S3a, fat stem cell acquisition;
S31a, collection fatty tissue;
Select step S2a reaches the patient of evaluation criteria, the fatty tissue at the abundant position of the fat contents such as patients abdomen, buttocks, thigh is aseptically extracted with liposuction machine, reject macroscopic blood vessel and fiber part in fat, by PBS fatty tissue 3 times, put into containing dual anti-(penicillin/streptomycin, be respectively 1% content) buffer in, gnotobasis saves backup under 4 DEG C of conditions.
S32a, obtain primary fat stem cell;
Take the fatty tissue obtained in step S31a, after shredding under aseptic condition, add 0.075% NTx enzyme, 37 DEG C of constant temperature also digest 40 minutes with 50rpm vibration velocity vibration shaking table, and the complete medium (DMEM in high glucose+8% hyclone+1% is dual anti-) being subsequently adding equivalent volumes stops digestion; Then, Digestive system is placed in ice bath, the Dnase I (deoxyribonuclease I) adding 10mg/L after blowing and beating 15 times with suction pipe hatches 15 minutes, filter through 100 micron screen, remove indigested fatty tissue and connective tissue, under 4 DEG C of environment, centrifugal 10 minutes of 1000rpm, mature fat cell is below the oil droplet of top layer, above culture medium, remove upper strata oil droplet, floating adipose cell is transferred in new centrifuge tube, adds PBS, centrifugal 10 minutes of 1000rpm in 4 DEG C of environment, repeat this centrifugal process for several times, namely obtain primary fat stem cell.
S33a, fat stem cell purification;
By the primary fat stem cell obtained in step S32a with 1% salt water washing 2��3 times to remove the erythrocyte of pollution, resuspended with complete medium, it is inoculated in the coated culture plate of poly-ornithine, cultivate 4 hours, remove supernatant, with the trypsinization of 0.25%, remove fibroblast, the endotheliocyte etc. that are difficult to digest, it is thus achieved that the primary fat stem cell after purification.
S34a, fat stem cell amplification cultivation;
Being placed in the cell culture bags of 300 milliliters by the primary fat stem cell after the purification obtained in step S33a, the inoculum density of primary fat stem cell is 1 �� 105Individual/ml, adds DMEM culture medium, is positioned over 37 DEG C, 5%CO2In incubator, saturated humidity is cultivated 8 days;
Wherein, DMEM culture medium is added with the ITS (insulin, transferrins, selenium) of 5% human serum, 10ng/ml, the streptomycin of 100U/ml and penicillin, EGF (the EpidermalGrowthFactor of 20ng/ml, epidermal growth factor), the LIF (leukemiainhibitoryfactor, leukaemia inhibitory factor) of 10ng/ml.
S4a, allograft skin substrate preparation;
Whole abdominal part of health pig and back skin Mechanical Method are carried out preliminary defat, hair is rejected clean with tweezers, first the subcutaneous tissue of Corii Sus domestica is removed with electric dermatome, remove epidermis 0.4mm again, make the skin corium skin graft that thickness is 0.3mm, after normal saline is cleaned, immerse sterilization in the medical bromogeramine solution that concentration is 0.1%, take out after 30 minutes, repeatedly rinse with physiological saline solution 10 times; Then skin graft is cut into 1cm �� 1cm size, PBS liquid rinses twice, it is soaked in the vial equipped with 0.25% trypsin, 0.02% ethanedioic acid ethylenediamine (EDTA) and 0.02% sodium hydroxide mixture slaking solution, digests 3d at 4 DEG C, rock about 5 times every day. Taking out skin graft after 4 days, PBS liquid rinses 2 times, puts into the vial equipped with 0.5%TritonX-100 solution, incubated at room temperature, and sustained oscillation 3d, until cell component is removed completely, it is thus achieved that acellular dermal.
Finally, rinse with the NaCl solution that mass fraction is 7% and the PBS liquid that pH value is 7.4; Wherein, the NaCl solution process time is 12h, and the phosphate buffered saline(PBS) process time is 12h, changes liquid every 6h, repeats 5 times, be stored in 4 DEG C of PBS liquid standby.
S5a, transdermal patches preparation;
S51a, allograft skin substrate sterilization processing;
Take out the allograft skin substrate obtained in step S4a to load between sterile purification in sterile aluminum foil film package bag, vacuum-pumping and sealing, 60Coradiation sterilizing 4 hours, irradiation dose 4kGy.
S52a, epoxy glue original solution and fat stem cell;
Take 0.2% collagen solution to add in centrifuge tube, use NaHCO2Adjust solution PH to 7.2, the fat stem cell obtained and collagen solution are quickly sufficiently mixed, make fat stem cell final concentration of 3 �� 10 in step S34a4Individual/ml.
S53a, the collagen gel containing fat stem cell is coated in allograft skin substrate;
Under aseptic condition, take the allograft skin substrate obtained in step S51a, the collagen gel containing fat stem cell obtained in step S52a is coated in allograft skin substrate, height about 2mm, room temperature standing 20min treats that it solidifies or puts incubator and treats collagen set, namely makes transdermal patches.
During use, transdermal patches is sticked on wound surface, sew up after alignment, pressure dressing and fixing; And for burn severe patient, multi-point injection fat stem cell can be adopted to coordinate the treatment of transdermal patches method, the volume of the fat stem cell of every some injecting step S34a acquisition is less than 250ml, and the density of fat stem cell is 3 �� 104Individual/ml.
Embodiment 2
It is different in that with embodiment 1, in the present embodiment,
S3b, fat stem cell acquisition;
S31b, collection fatty tissue;
Select step S2a reaches the patient of evaluation criteria, the fatty tissue at the abundant position of the fat contents such as patients abdomen, buttocks, thigh is aseptically extracted with liposuction machine, reject macroscopic blood vessel and fiber part in fat, by PBS fatty tissue 3 times, put into containing dual anti-(penicillin/streptomycin, be respectively 1% content) buffer in, gnotobasis saves backup under 4 DEG C of conditions.
S32b, obtain primary fat stem cell;
Take the fatty tissue obtained in step S31b, after shredding under aseptic condition, add 0.075% NTx enzyme, 37 DEG C of constant temperature also digest 20 minutes with 50rpm vibration velocity vibration shaking table, and the complete medium (DMEM in high glucose+5% hyclone+2% is dual anti-) being subsequently adding equivalent volumes stops digestion; Then, Digestive system is placed in ice bath, the Dnbse I (deoxyribonuclease I) adding 10mg/L after blowing and beating 15 times with suction pipe hatches 20 minutes, filter through 100 micron screen, remove indigested fatty tissue and connective tissue, under 4 DEG C of environment, centrifugal 10 minutes of 1000rpm, mature fat cell is below the oil droplet of top layer, above culture medium, remove upper strata oil droplet, floating adipose cell is transferred in new centrifuge tube, adds PBS, centrifugal 10 minutes of 1000rpm in 4 DEG C of environment, repeat this centrifugal process for several times, namely obtain primary fat stem cell.
S33b, fat stem cell purification;
By the primary fat stem cell obtained in step S32b with 1% salt water washing 3 times to remove the erythrocyte of pollution, resuspended with complete medium, it is inoculated in the coated culture plate of poly-ornithine, cultivate 2 hours, remove supernatant, with the trypsinization of 0.25%, remove fibroblast, the endotheliocyte etc. that are difficult to digest, it is thus achieved that the primary fat stem cell after purification.
S34b, fat stem cell amplification cultivation;
Being placed in the cell culture bags of 300 milliliters by the primary fat stem cell after the purification obtained in step S33b, the inoculum density of primary fat stem cell is 0.5 �� 105Individual/ml, adds DMEM culture medium, is positioned over 37 DEG C, 5%CO2In incubator, saturated humidity is cultivated 7 days;
Wherein, DMEM culture medium is added with the ITS (insulin, transferrins, selenium) of 3% human serum, 5ng/ml, the streptomycin of 50U/ml and the penicillin of 50U/ml, EGF (the EpidermblGrowthFbctor of 5ng/ml, epidermal growth factor), the LIF (leukemibinhibitoryfbctor, leukaemia inhibitory factor) of 5ng/ml.
S4b, allograft skin substrate preparation;
Whole abdominal part of health pig and back skin Mechanical Method are carried out preliminary defat, hair is rejected clean with tweezers, first the subcutaneous tissue of Corii Sus domestica is removed with electric dermatome, remove epidermis 0.2mm again, make the skin corium skin graft that thickness is 0.2mm, after normal saline is cleaned, immerse sterilization in the medical bromogeramine solution that concentration is 0.1%, take out after 20 minutes, repeatedly rinse with physiological saline solution 10 times; Then skin graft is cut into 1cm �� 1cm size, PBS liquid rinses twice, it is soaked in the vial equipped with 0.10% trypsin, 0.01% ethanedioic acid ethylenediamine (EDTB) and 0.01% sodium hydroxide mixture slaking solution, digests 3d at 4 DEG C, rock about 5 times every day. Taking out skin graft after 4 days, PBS liquid rinses 2 times, puts into the vial equipped with 0.4%TritonX-100 solution, incubated at room temperature, and sustained oscillation 2d, until cell component is removed completely, it is thus achieved that acellular dermal.
Finally, rinse with the NaCl solution that mass fraction is 5% and the PBS liquid that pH value is 7.4; Wherein, the NbCl solution-treated time is 8h, and the phosphate buffered saline(PBS) process time is 8h, changes liquid every 6h, repeats 5 times, be stored in 4 DEG C of PBS liquid standby.
S5b, transdermal patches preparation;
S51b, allograft skin substrate sterilization processing;
Take out the allograft skin substrate obtained in step S4b to load between sterile purification in sterile aluminum foil film package bag, vacuum-pumping and sealing, 60Coradiation sterilizing 3 hours, irradiation dose 3kGy.
S52b, epoxy glue original solution and fat stem cell;
Take 0.1% collagen solution to add in centrifuge tube, use NaHCO2Adjust solution PH to 7.2, the fat stem cell obtained and collagen solution are quickly sufficiently mixed, make fat stem cell final concentration of 2 �� 10 in step S34b4Individual/ml.
S53b, the collagen gel containing fat stem cell is coated in allograft skin substrate;
Under aseptic condition, take the allograft skin substrate obtained in step S51b, the collagen gel containing fat stem cell obtained in step S52b is coated in allograft skin substrate, height about 3mm, room temperature standing 30min treats that it solidifies or puts incubator and treats collagen set, namely makes transdermal patches.
During use, transdermal patches is sticked on wound surface, sew up after alignment, pressure dressing and fixing; And for burn severe patient, multi-point injection fat stem cell can be adopted to coordinate the treatment of transdermal patches method, the volume of the fat stem cell of every some injecting step S34b acquisition is less than 250ml, and the density of fat stem cell is 2 �� 104Individual/ml.
Embodiment 3
It is different in that with embodiment 1, in the present embodiment,
S3c, fat stem cell acquisition;
S31c, collection fatty tissue;
Select step S2a reaches the patient of evaluation criteria, the fatty tissue of patients abdomen is aseptically extracted with liposuction machine, reject macroscopic blood vessel and fiber part in fat, by PBS fatty tissue 3 times, put into containing dual anti-(penicillin/streptomycin, be respectively 1% content) buffer in, gnotobasis saves backup under 4 DEG C of conditions.
Wherein, this patient is deep?degreeburn, and arm part burn surface area is 100cm2Left and right.
S32c, obtain primary fat stem cell;
Take the fatty tissue obtained in step S31c, after shredding under aseptic condition, add 0.075% NTx enzyme, 37 DEG C of constant temperature also digest 60 minutes with 50rpm vibration velocity vibration shaking table, and the complete medium (DMEM in high glucose+10% hyclone+0.5% is dual anti-) being subsequently adding equivalent volumes stops digestion; Then, Digestive system is placed in ice bath, the Dncse I (deoxyribonuclease I) adding 10mg/L after blowing and beating 15 times with suction pipe hatches 20 minutes, filter through 100 micron screen, remove indigested fatty tissue and connective tissue, under 4 DEG C of environment, centrifugal 10 minutes of 1000rpm, mature fat cell is below the oil droplet of top layer, above culture medium, remove upper strata oil droplet, floating adipose cell is transferred in new centrifuge tube, adds PCS and clean, centrifugal 10 minutes of 1000rpm in 4 DEG C of environment, repeat this centrifugal process for several times, namely obtain primary fat stem cell.
S33c, fat stem cell purification;
By the primary fat stem cell obtained in step S32c with 1% salt water washing 3 times to remove the erythrocyte of pollution, resuspended with complete medium, it is inoculated in the coated culture plate of poly-ornithine, cultivate 6 hours, remove supernatant, with the trypsinization of 0.25%, remove fibroblast, the endotheliocyte etc. that are difficult to digest, it is thus achieved that the primary fat stem cell after purification.
S34c, fat stem cell amplification cultivation;
Being placed in the cell culture bags of 300 milliliters by the primary fat stem cell after the purification obtained in step S33c, the inoculum density of primary fat stem cell is 2 �� 105Individual/ml, adds DMEM culture medium, is positioned over 37 DEG C, 5%CO2In incubator, saturated humidity is cultivated 10 days;
Wherein, DMEM culture medium is added with the ITS (insulin, transferrins, selenium) of 2% human serum, 15ng/ml, the streptomycin of 150U/ml and the penicillin of 150U/ml, EGF (the EpidermclGrowthFcctor of 35ng/ml, epidermal growth factor), the LIF (leukemicinhicitoryfcctor, leukaemia inhibitory factor) of 15ng/ml.
S4c, allograft skin substrate preparation;
Whole abdominal part of health pig and back skin Mechanical Method are carried out preliminary defat, hair is rejected clean with tweezers, first the subcutaneous tissue of Corii Sus domestica is removed with electric dermatome, remove epidermis 0.6mm again, make the skin corium skin graft that thickness is 0.4mm, after normal saline is cleaned, immerse sterilization in the medical bromogeramine solution that concentration is 0.1%, take out after 40 minutes, repeatedly rinse with physiological saline solution 10 times; Then skin graft is cut into 1cm �� 1cm size, PBS liquid rinses twice, it is soaked in the vial equipped with 0.40% trypsin, 0.03% ethanedioic acid ethylenediamine (EDTC) and 0.03% sodium hydroxide mixture slaking solution, digests 2d at 4 DEG C, rock about 5 times every day. Taking out skin graft after 4 days, PBS liquid rinses 2 times, puts into the vial equipped with 0.6%TritonX-100 solution, incubated at room temperature, and sustained oscillation 4d, until cell component is removed completely, it is thus achieved that acellular dermal.
Finally, rinse with the NaCl solution that mass fraction is 9% and the PBS liquid that pH value is 7.4; Wherein, the NaCl solution process time is 16h, and the phosphate buffered saline(PBS) process time is 16h, changes liquid every 6h, repeats 5 times, be stored in 4 DEG C of PBS liquid standby.
S5c, transdermal patches preparation;
S51c, allograft skin substrate sterilization processing;
Take out the allograft skin substrate obtained in step S4c to load between sterile purification in sterile aluminum foil film package bag, vacuum-pumping and sealing, 60Coradiation sterilizing 5 hours, irradiation dose 5kGy.
S52c, epoxy glue original solution and fat stem cell;
Take 0.3% collagen solution to add in centrifuge tube, use NaHCO2Adjust solution PH to 7.2, the fat stem cell obtained and collagen solution are quickly sufficiently mixed, make fat stem cell final concentration of 4 �� 10 in step S34c4Individual/ml.
S53c, the collagen gel containing fat stem cell is coated in allograft skin substrate;
Under aseptic condition, take the allograft skin substrate obtained in step S51c, the collagen gel containing fat stem cell obtained in step S52c is coated in allograft skin substrate, height about 1mm, room temperature standing 10min treats that it solidifies or puts incubator and treats collagen set, namely makes transdermal patches.
Before patient's burned part paster, localized burn wound surface need to be made conventional debridement treatment, remove dirt and the epithelium that comes off, remove blister and sterilize, the transdermal patches prepared being built according to burned part area and shape, then by 130cm in the present embodiment2The transdermal patches of left and right, facing to burn wound, makes paster and wound surface be adjacent to and cover whole wound surface, and paster is more than edge of wound about 2��3cm. Opening gauze after 5d, observe wound surface, it has been found that wound surface oozes out less, the transdermal patches more renewed, when changing paster, pain is substantially lighter than the allograft skin paster not using autologous fat stem cell to smear. Again every 5d, open gauze, it has been found that damage location starts epithelization occur, without infecting, the paster again more renewed. Until 21d, opening gauze, wound surface heals substantially. After healing, wound surface is followed up a case by regular visits to, and healing wound surface scar hyperplasia is few, and pigmentation is few. And prior art adopts allograft skin substrate repair burned skin and need 30 days even longer reparation phases, longer relative to the transdermal patches required time of the present invention, and it is easier to more by force cause inflammation due to immunologic rejection, it is easy to cause pigment to accumulate.
It addition, the patient of transdermal patches adopting the present embodiment to provide is detected respectively its before the treatment and after treatment the 7th and 21d blood in Immunoglobulin IgA, IgG, IgM content, testing result is table 1 below such as:
Table 1
(wherein, matched group is the content of Immunoglobulin IgA, IgG and IgM in normal human blood. )
Testing result: by adopting transdermal patches provided by the invention treatment skin burn wound, treatment the 7th day, immunoglobulin content was significantly raised; When treating the 20th day, immunoglobulin content recovers normally, thus to illustrate that transdermal patches provided by the invention not only shortens the skin repair time, and repairing effect is better substantially.
Above embodiments of the invention are described; but the invention is not limited in above-mentioned detailed description of the invention; above-mentioned detailed description of the invention is merely schematic; rather than it is restrictive; those of ordinary skill in the art is under the enlightenment of the present invention; without departing under present inventive concept and scope of the claimed protection situation, it may also be made that a lot of form, these belong within the protection of the present invention.
Claims (10)
1. the preparation method for treating the transdermal patches of skin burn, it is characterised in that comprise the following steps:
Obtain fat stem cell and allograft skin substrate respectively;
Described allograft skin substrate it is inoculated in collagen solution after being mixed by described fat stem cell;
Wherein, described fat stem cell derives from skin burn autologous patient.
2. the preparation method of the transdermal patches for treating skin burn according to claim 1, it is characterised in that final concentration of (2��4) �� 10 of described fat stem cell4Individual/ml.
3. the preparation method of the transdermal patches for treating skin burn according to claim 1, it is characterised in that in described collagen solution, the mass fraction of collagen is 0.1��0.3%.
4. the preparation method of the transdermal patches for treating skin burn according to claim 2, it is characterised in that the acquisition process of described fat stem cell, comprises the following steps:
Under aseptic condition, take the fatty tissue of described skin burn patient, reject macroscopic blood vessel and fiber part in fat, after PBS solution is cleaned, shred, enzymolysis vibration digestion 20��60 minutes, stop digestion with complete medium, then be placed in ice bath and blow and beat digestion 10��20 minutes, filter, after removing indigested fatty tissue and connective tissue, filtrate is centrifuged, removes upper strata oil droplet, take off a layer adipose cell, after PBS, namely obtain primary fat stem cell.
5. the preparation method of the transdermal patches for treating skin burn according to claim 4, it is characterised in that the acquisition process of described fat stem cell is further comprising the steps of:
Wash described primary fat stem cell, remove erythrocyte, resuspended with complete medium, then it is inoculated on the coated culture plate of poly-ornithine, cultivate 2��6 hours, remove trypsinization after supernatant, remove indigested fibroblast and endotheliocyte, namely obtain the primary fat stem cell after purification.
6. the preparation method of the transdermal patches for treating skin burn according to claim 5, it is characterised in that the acquisition process of described fat stem cell is further comprising the steps of:
Take the primary fat stem cell after purification with 0.5��2 �� 105The density of individual/ml is inoculated in DMEM culture medium and carries out amplification cultivation 7��10 days, changes liquid every 2-3 days once.
7. the preparation method of the transdermal patches for treating skin burn according to claim 6, it is characterized in that, described DMEM culture medium is added with the LIF of EGF and the 5��15ng/ml of 2��5% human serums, the ITS of 5��15ng/ml, the streptomycin of 50��150U/ml, 50��150U/ml penicillin, 5��35ng/ml.
8. the preparation method of the transdermal patches for treating skin burn according to claim 1, it is characterised in that the acquisition process of described allograft skin substrate, comprises the following steps:
Take health pig abdominal part or back skin carries out defat depilation process, remove the epidermis of 0.2��0.6mm, make the skin corium skin graft that thickness is 0.2��0.4mm, sterilize 20��40 minutes after cleaning, repeatedly clean, digest 2��4 days in Digestive system, repeatedly clean skin graft, be subsequently adding nonionic surfactant and hatch 2��4 days, remove completely to cell component, obtain acellular dermal, then repeatedly rinse with sodium chloride and PBS respectively, namely obtain allograft skin substrate.
9. the preparation method of the transdermal patches for treating skin burn according to claim 8, it is characterized in that, including mass percent in described Digestive system is 0.10��0.40% trypsin, 0.01��0.03% ethanedioic acid ethylenediamine and 0.01��0.03% sodium hydroxide.
10. the transdermal patches being used for treating skin burn, it is characterised in that the transdermal patches preparation method for treating skin burn described in any one of claim 1-9 obtains.
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| CN111893085A (en) * | 2020-08-04 | 2020-11-06 | 北京臻溪谷医学研究中心（有限合伙） | Method for obtaining artificial skin through stem cell in-vitro differentiation and application thereof |
| CN111888526A (en) * | 2020-06-16 | 2020-11-06 | 陈翔 | Skin sheet for mixed transplantation of heterogeneous species |
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